Implementation of HIV Drug Resistance Testing in Kenya

Background: Testing for HIV drug resistance prior to antiretroviral therapy (ART) is standard of care in the United States but is rarely performed in Africa due to its high cost. The prevalence of HIV drug resistance is increasing in resource-limited settings (RLS). An affordable and feasible strategy is needed in RLS to identify individuals with drug resistance and prescribe effective ART. Our group developed a low-cost oligonucleotide ligation assay (OLA) that detects mutations conferring resistance. Little data exists on implementing HIV drug resistance testing in RLS. Here, we describe the implementation of OLA in one laboratory in Kenya and discuss practical aspects of executing the OLA over two years. Methods: The OLA was transferred to a research laboratory at the Coptic Hope Center in Nairobi, Kenya, as part of a randomized trial testing use of the OLA in individuals starting first-line ART to improve virologic outcome. The Seattle Lab Manager (SLM) transported equipment needed for OLA to Nairobi, set up the laboratory, and trained two Kenyan lab technicians to perform OLA. Two additional technicians were later trained. Technicians had education either as a lab technologist or lab scientist, with limited training in molecular techniques. Results: OLA was successfully performed by Kenyan lab technicians on 565 blood samples. Each week, OLA was performed on approximately 7 samples, requiring an estimated 10 hours of technician labor, and 2 hours of remote technical support, review of test results and oversight from the SLM. Some sample results were delayed during two temporary, month-long pauses in testing of specimens, due to suboptimal performance of the OLA. This required trouble-shooting by the SLM in conjunction with lab personnel. Conclusion: OLA technology was successfully transferred to the Kenyan laboratory. However, it required time-intensive technician labor and substantial oversight by the SLM. The complexity of OLA, and a paucity of lab technicians and on-site supervisors trained in molecular techniques are potential bottlenecks for implementation of the current version of OLA at a larger population-level. Research is ongoing to develop OLA Simple, a simplified kit aimed to address these challenges and serve as a point-of-care assay

P16 expression and recurrent cervical intraepithelial neoplasia after cryotherapy among women living with HIV

Background: The expression of p16 protein, a surrogate marker for high-risk human papillomavirus (hrHPV), is associated with cervical dysplasia. We evaluated correlates of p16 expression at treatment for high-grade cervical lesions and its utility in predicting the recurrence of cervical intraepithelial lesions grade 2 or higher (CIN2+) following cryotherapy among women with HIV. Methods: This is a subgroup analysis of women with HIV in Kenya with baseline cervical biopsy-confirmed CIN2+ who were randomized to receive cryotherapy and followed every six-months for two-years for biopsy-confirmed recurrence of CIN2+. P16 immunohistochemistry was performed on the baseline cervical biopsy with a positive result defined as strong abnormal nuclear expression in a continuous block segment of cells (at least 10–20 cells). Results: Among the 200 women with CIN2+ randomized to cryotherapy, 160 (80%) had a baseline cervical biopsy specimen available, of whom 94 (59%) were p16-positive. p16 expression at baseline was associated with presence of any one of 14 hrHPV genotypes [Odds Ratio (OR)  =  3.2; 95% Confidence Interval (CI), 1.03–9.78], multiple lifetime sexual partners (OR  =  1.6; 95% CI, 1.03–2.54) and detectable plasma HIV viral load (\u3e1,000 copies/mL; OR  =  1.43; 95% CI, 1.01– 2.03). Longer antiretroviral therapy duration (≥2  years) at baseline had lower odds of p16 expression (OR  =  0.46; 95% CI, 0.24–0.87) than \u3c2  years of antiretroviral therapy. Fifty-one women had CIN2+ recurrence over 2-years, of whom 33 (65%) were p16-positive at baseline. p16 was not associated with CIN2+ recurrence (Hazard Ratio  =  1.35; 95% CI, 0.76–2.40). Conclusion: In this population of women with HIV and CIN2+, 41% of lesions were p16 negative and baseline p16 expression did not predict recurrence of cervical neoplasia during two-year follow up

Partner Disclosure and Early CD4 Response among HIV-Infected Adults Initiating Antiretroviral Treatment in Nairobi Kenya

Background: Disclosure of HIV serostatus can have significant benefits for people living with HIV/AIDS. However, there is limited data on whether partner disclosure influences ART treatment response. Methods: We conducted a retrospective cohort study of newly diagnosed, ART-naïve HIV-infected adults (\u3e18 years) who enrolled at the Coptic Hope Center in Nairobi, Kenya between January 1st2009 and July 1st 2011 and initiated ART within 3 months. Analysis was restricted to adults who reported to have either disclosed or not disclosed their HIV status to their partner. Analysis of CD4 response at 6 and 12 months post-ART was stratified by age group. Results: Among 615 adults newly initiating ART with partner disclosure data and 12 month follow-up, mean age was 38 years and 52% were male; 76% reported that they had disclosed their HIV-status to their partner. Those who disclosed were significantly younger and more likely to be married/cohabitating than non-disclosers. At baseline, median CD4 counts were similar between disclosure groups. Among younger adults (\u3c 38 years) those who disclosed had higher CD4 recovery than those who did not at 6 months post- ART (mean difference = 31, 95% CI 3 to 58 p = 0.03) but not at 12 months (mean difference = 17, 95% CI -19 to 52, p = 0.4). Among older adults (≥ 38years) there was no observed difference in CD4 recovery at 6 or 12 months between disclosure groups. Conclusion: Among younger adults, disclosure of HIV status to partners may be associated with CD4 recovery following ART

Residual disease and HPV persistence after cryotherapy for cervical intraepithelial neoplasia grade 2/3 in HIV positive women in Kenya

Objective: To assess residual cervical intraepithelial neoplasia (CIN) 2/3 disease and clearance of high-risk (hr) human papillomavirus (HPV) infections at 6 months after cryotherapy among HIV-positive women. Design: Follow-up study. Methods: 79 HIV-positive women received cryotherapy for CIN2/3 in Nairobi, Kenya, and underwent conventional cytology 6 months later. Biopsies were performed on high grade cytological lesions and hrHPV was assessed before (cervical cells and biopsy) and after cryotherapy (cells). Results: At 6 months after cryotherapy CIN2/3 had been eliminated in 61 women (77.2%; 95% Confidence Interval, (CI):66.4–85.9). 18 women (22.8%) had residual CIN2/3, and all these women had hrHPV at baseline. CD4 count and duration of combination antiretroviral therapy (cART) were not associated with residual CIN2/3. CIN3 instead of CIN2 was the only significant risk factor for residual disease (odds ratio, OR vs CIN2 = 4.3; 95% CI: 1.2–15.0) among hrHPV-positive women after adjustment for age and HPV16 infection. Persistence of hrHPV types previously detected in biopsies was found in 77.5% of women and was associated with residual CIN2/3 (OR = 8.1, 95% CI: 0.9–70). The sensitivity, specificity, and negative predictive value of hrHPV test in detecting residual CIN2/3 were 0.94, 0.36, and 0.96 respectively. Conclusions: Nearly one quarter of HIV-positive women had residual CIN2/3 disease at 6 months after cryotherapy, and the majority had persistent hrHPV. CD4 count and cART use were not associated with residual disease or hrHPV persistence. The value of hrHPV testing in the detection of residual CIN2/3 was hampered by a low specificity

Oligonucleotide ligation assay detects HIV drug resistance associated with virologic failure among antiretroviral-naive adults in Kenya

Background: Transmitted drug resistance (TDR) is increasing in some areas of Africa. Detection of TDR may predict virologic failure of first-line non-nucleoside reverse-transcriptase inhibitor (NNRTI)-based antiretroviral therapy (ART). We evaluated the utility of a relatively inexpensive oligonucleotide ligation assay (OLA) to detect clinically relevant TDR at time of ART initiation. Methods: Pre-ART plasmas from ART-naive Kenyans initiating an NNRTI-based fixed-dose combination ART in a randomized adherence trial conducted in 2006 were retrospectively analyzed by OLA for mutations conferring resistance to NNRTI (K103N, Y181C, and G190A) and lamivudine (M184V). Post-ART plasmas were analyzed for virologic failure (≥1,000 copies/mL) at 6 month intervals over 18-month follow-up. Pre-ART plasmas of those with virologic failure were evaluated for drug resistance by consensus and 454-pyrosequencing. Results: Among 386 participants, TDR was detected by OLA in 3.89% [95% Confidence Interval (CI), 2.19-6.33], and was associated with a 10-fold higher rate of virologic failure [Hazard Ratio (HR), 10.39; 95% CI, 3.23-32.41; p Conclusions: Detection of TDR by a point mutation assay may prevent use of sub-optimal ART

Increasing HIV-1 pretreatment drug resistance among antiretroviral-naïve adults initiating treatment between 2006 and 2014 in Nairobi, Kenya

Antiretroviral-naïve adults initiating antiretroviral therapy in Nairobi, Kenya were tested for HIV-1 drug resistance at codons K103N, Y181C, G190A, M184V, and K65R using an oligonucleotide ligation assay. Prevalence of pretreatment drug resistance increased from 3.89% in 2006 to 10.93% in 2014 (P \u3c 0.001), and 95% of those with resistance had at least one nonnucleoside reverse transcriptase inhibitor mutation. Resistance to tenofovir (K65R) was found in 2014 but not in 2006

Cervical HIV-1 RNA shedding after cryotherapy among HIV-positive women with cervical intraepithelial neoplasia stage 2 or 3

Objective: To determine the effect of cryotherapy on HIV-1 cervical shedding. Design: Prospective cohort study. Methods: Five hundred HIV-positive women enrolled at an HIV treatment clinic in Nairobi, Kenya were screened for cervical cancer. Women diagnosed with cervical intraepithelial neoplasia stage 2 or 3 (CIN 2/3) by histology were offered cryotherapy treatment. The first 50 women had cervical swabs taken at baseline and at 2 and 4 weeks following treatment. Swabs were analyzed for HIV-1 RNA and compared using General Estimating Equation (GEE) with binomial or Gaussian links. Results: Of the 50 women enrolled, 40 were receiving antiretroviral therapy (ART) and 10 were not receiving ART at the time of cryotherapy and during study follow-up. Among all women, the odds of detectable cervical HIV-1 RNA did not increase at 2 weeks [odds ratio (OR) 1.18; 95% confidence interval (CI) 0.65-2.13] or 4 weeks (OR 1.29; 95% CI 0.71-2.33) following cryotherapy. Among 10 women not receiving ART, the OR of detectable shedding at 2 weeks was higher, but not statistically significant (OR 4.02; 95% CI 0.53-30.79; P = 0.2), and at 4 weeks remained unchanged (OR 1.00; 95% CI 0.27-3.74). Conclusion: There was no increase in detectable cervical HIV-1 RNA among HIV-positive women after cryotherapy. The risk of HIV-1 transmission after cryotherapy may not be significant, particularly among women already on ART at the time of cervical treatment. However, further investigation is needed among women not receiving ART

Comparison of HPV DNA testing in cervical exfoliated cells and tissue biopsies among HIV-positive women in Kenya

HIV-positive women are infected with human papillomavirus (HPV) (especially with multiple types), and develop cervical intraepithelial neoplasia (CIN) and cervical cancer more frequently than HIV-negative women. We compared HPV DNA prevalence obtained using a GP5+/6+ PCR assay in cervical exfoliated cells to that in biopsies among 468 HIV-positive women from Nairobi, Kenya. HPV prevalence was higher in cells than biopsies and the difference was greatest in 94 women with a combination normal cytology/normal biopsy (prevalence ratio, PR = 3.7; 95% confidence interval, CI: 2.4-5.7). PR diminished with the increase in lesion severity (PR in 58 women with high-grade squamous intraepithelial lesions (HSIL)/CIN2-3 = 1.1; 95% CI: 1.0-1.2). When HPV-positive, cells contained 2.0- to 4.6-fold more multiple infections than biopsies. Complete or partial agreement between cells and biopsies in the detection of individual HPV types was found in 91% of double HPV-positive pairs. The attribution of CIN2/3 to HPV16 and/or 18 would decrease from 37.6%, when the presence of these types in either cells or biopsies was counted, to 20.2% when it was based on the presence of HPV16 and/or 18 (and no other types) in biopsies. In conclusion, testing HPV on biopsies instead of cells results in decreased detection but not elimination of multiple infections in HIV-positive women. The proportion of CIN2/3 attributable to HPV16 and/or 18 among HIV-positive women, which already appeared to be lower than that in HIV-negative, would then further decrease. The meaning of HPV detection in cells and random biopsy from HIV-positive women with no cervical abnormalities remains unclear. What\u27s new? Assignment of human papillomavirus (HPV) types to individual cervical lesions is essential for the understanding of the biology of different HPV types, efficacy of HPV vaccines, and design of detection assays. Such attribution is however hampered in HIV-positive women by the high proportion of multiple HPV infections. This study is the first to systematically compare HPV detection in paired cervical exfoliated cells and cervical tissue biopsies. HPV testing using biopsies instead of cells results in decreased detection of multiple infections in HIV-positive women. Exclusive reliance on biopsies also decreased the proportion of CIN2/3 attributable to vaccine-preventable HPV16 and/or 18 infection

Increased Cervical Human Immunodeficiency Virus (HIV) RNA Shedding Among HIV-Infected Women Randomized to Loop Electrosurgical Excision Procedure Compared to Cryotherapy for Cervical Intraepithelial Neoplasia 2/3

Background Treatment of human immunodeficiency virus (HIV)–infected women to prevent cervical cancer may stimulate HIV RNA cervical shedding and risk HIV transmission. Methods From 2011 to 2014, 400 HIV-infected women diagnosed with cervical intraepithelial neoplasia 2/3 in Kenya were randomized to loop electrosurgical excision procedure (LEEP) or cryotherapy. Cervical samples were collected at baseline and 3 weekly intervals. Samples were tested for HIV RNA using the Gen-Probe Aptima HIV assay with a minimum detection level of 60 copies/swab and analyzed using generalized estimating equations. Results Women who received LEEP had significantly higher cervical HIV RNA levels than those who received cryotherapy at weeks 2 (adjusted incident rate ratio [aIRR], 1.07; P = .038) and 3 (aIRR, 1.08; P = .046). Within LEEP, significantly higher cervical shedding was found at weeks 2 (2.03 log10 copies/swab; P < .001) and 3 (2.04 log10 copies/swab; P < .001) compared to baseline (1.80 log10 copies/swab). Cervical HIV RNA was significantly higher following LEEP for up to 3 weeks among women on antiretroviral treatment (ART) (0.18 log10 copies/swab increase; P = .003) and in ART-naive women (1.13 log10 copies/swab increase; P < .001) compared to baseline. Within cryotherapy, cervical shedding increased in ART-naive women (0.72 log10 copies/swab increase; P = 0.004) but did not increase in women on ART. Conclusions Women randomized to LEEP had a larger increase in post-procedural cervical HIV shedding than cryotherapy. Benefits of cervical cancer prevention outweigh the risk of HIV sexual transmission; our findings underscore the importance of risk-reduction counseling

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London